Transplantation of stem cells is a preferred strategy in the treatment of a variety of hematological malignancies and disorders. In recent years, the use of peripheral blood as a source of hematopoietic stem and progenitor cells (HSPC) for transplantation after a high-dose chemotherapy has emerged as a common clinical practice. Successful transplantation requires the infusion of a sufficient number of HSPC capable of homing and engraftment to the bone marrow (BM), and regenerating a full array of hematopoietic cell lineages. Under steady-state conditions, there are very low frequencies of circulating HPSC. However, these low levels can be amplified by a process termed HSPC mobilization. Thus, elevating the number of HSPC mobilized to the blood will improve the yield of cell collection for transplantation and will also have the potential to shorten recovery from cytopenia and reduce morbidity and mortality.
The cytokine granulocyte colony-stimulating factor (G-CSF) is so far the major mobilizing agent inducing a dramatic elevation in the number of HSPC in the blood (Lapidot, T. & Petit, I., 2002). However, G-CSF therapy is very expensive and requires a 5 day repeated dosing, and its efficacy in HSPC mobilization greatly varies among different patients. In addition, G-CSF therapy is frequently associated with side effects such as bone pain, headache and myalgia (Tigue, C. C., et al., 2007). Hence, improved methods to mobilize peripheral blood HSPC for hematopoietic rescue are warranted.
Evidence suggests that interactions between the chemokine stromal-derived factor-1 (SDF-1, also named CXCL12) and its major receptor CXCR4 play a crucial role in HPSC mobilization, homing, engraftment, survival and retention within the BM (Dar, A., Kollet, O. & Lapidot, T., 2006). Thus, disruption of SDF-1/CXCR4 interactions may result in mobilization of HSPC. Indeed, blocking of CXCR4 with its specific antagonist AMD3100 results in the rapid mobilization of CD34+ hematopoietic progenitor cells (HPC) from the human BM to the blood (Broxmeyer, H. E., et al., 2005, Liles, W. C., et al., 2005, Dvine et al., 2008). In addition, combining AMD3100 with G-CSF has produced an additive effect (Gazitt, Y., Freytes, C. O., Akay, C., Badel, K. & Calandra, G., 2007). U.S. Pat. No. 6,365,583 further discloses a method to treat a subject who would be benefited by elevation of white blood cell count which method comprises administering to said subject a cyclic polyamine such as AMD3100. However, a clinical trial with AMD3100 in HIV-infected individuals was halted due to its cardio-toxic effect (Hendrix, C. W., et al., 2004).
A strong and rapid mobilization effect was demonstrated in the murine system by using another CXCR4 antagonist, 4F-benzoyl-TN14003, that provided also an additive effect with G-CSF (see, for example, Abraham, M., et al., 2007 and WO 2008/075369). Mobilization of human HSPC by 4F-benzoyl-TN14003 has not been clinically tested. Various other agents have been suggested to be involved in modulating CXCR4/SDF-1 interactions and are being evaluated for their therapeutic potential in affecting different functions and processes mediated by hematopoietic cells, including HSPC mobilization. For example, U.S. Pat. No. 7,435,718 is directed to SDF-1 analogs having CXCR4 antagonistic activity useful in the treatment of hematopoietic cells in vitro and in vivo, for example in increasing the rate of hematopoietic stem or progenitor cellular multiplication, self-renewal, expansion, proliferation, or peripheralization. WO 01/85196 suggests that hematopoietic cell proliferation may be modulated by ex vivo exposure to CXCR4 antagonists derived from SDF-1 (P2G), in which glycine is substituted for proline at amino acid position 2.
Other such agents are described, for example, in U.S. Pat. No. 7,291,631 and PCT Pub. No. WO 01/85196 and by Zeng et al. (2008), Tavor et al. (2004), Tamamura et al. (2005) and Burger and Peled (2009). U.S. Pat. Appl. Pub. No. 2004/0209921 discloses heterocyclic compounds that bind to chemokine receptors, including CXCR4 and CCR5, which may possess protective effects against infection of target cells by a human immunodeficiency virus (HIV). Other potential uses for these compounds suggested by '921 are enhancing the population of progenitor and/or stem cells, stimulating the production of white blood cells, and/or effecting regeneration of cardiac tissue.
Other publications relate to the use of CXCR4 antagonists in cancer therapy. For example, WO 2009140124 is directed to antibodies that bind human CXCR4 and are characterized as having high affinity and strong neutralizing properties, disclosed to be useful in the treatment of tumor growth, invasion, angiogenesis and metastasis.
PCT Pub. No. WO 2007/074456, to some of the inventors of the present invention, is directed to phenylalanine, cysteine, derivatives of said amino acids, peptides comprising them, and to their use in diseases, disorders or conditions whose pathology is caused by or associated with CXCR4 activity and/or cell motility, such as cancer, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis.
It has been suggested that certain CXCR4 mediated activities may also be affected by a defensin peptide, namely human β-defensin-3 (HBD3). Defensins are the members of antimicrobial peptides (AMPs) family employed as an innate immunity nonspecific defensive mechanism. Shortly after microbial infection, the AMPs are released and rapidly mobilized to neutralize a broad range of microorganisms (de Leeuw, E. et al., 2007). Being highly positively charged and disulfide-bonded, defensins bind to negatively charged bacterial membrane targets including LPS, polysaccharides, teicholic acids and phopspolipids. Membrane depolarization and permeabilization appear to be the predominant mechanisms of action of antimicrobial defensins (Schneider, J. J. et al., 2005). The presence of phosphatidilcholines in cytoplasmic membranes of eukaryotic cells and its electrostatic effects may account, at least in part, for the selectivity of defensins to bacteria and the relatively lower toxicity to eukaryotic cells (Nishimura, M., et al., 2004). So far, however, intracellular processing, storage, and release pathways of the defensins remain to be defined.
HBD3 is a 45 amino acids cysteine-enriched and highly cationic peptide, characterized by an exceptionally high net charge (+11), a broad spectrum of antimicrobial activity and relatively low cytotoxicity. It is expressed by epithelial cells, monocytes, dendritic cells and keratinocytes, and its expression is induced by IL-1, TNF-α, IFN-γ as well as by growth factors and various bacteria and viruses, whereas corticosteroids and cysteine proteases Cathepsins B, L, and S degrade and inactivate HBD3 (Dhople, V., Krukemeyer, A. & Ramamoorthy, A., 2006).
Apart from the antibacterial activity, HBD3 has been reported to inhibit HIV entry to the CXCR4 expressing target cells, and to internalize CXCR4 and decrease SDF-1-induced chemotaxis of T cells (Feng, Z. et al., 2006). HBD3 also increases migration of monocytes and CCR6-transfected human embryonic kidney HEK-293 cells (Wu, Z., et al., 2003). By direct binding to CCR6, HBD3 induces secretion of the proinflammatory cytokine IL-18 in human keratinocytes (Niyonsaba, F. et al., 2005) although participation of CCR6 as a possible receptor for HBD3 is doubtful (Soruri, A. et al., 2007). In addition, HBD3 induces the secretion of tissue remodeling proteins such as matrix metalloproteinases (MMP-1 and MMP-13) and reduces the levels of MMPs' inhibitors (TIMP-1/-2) in human cartilage (Varoga, D., et al., 2005). More recently it has been shown that HBD3 can induce expression of the co-stimulatory molecules CD80, CD86, and CD40 on monocytes and myeloid dendritic cells in a toll-like receptor (TLR)-dependent manner (Funderburg, N., et al., 2007). PCT Pub. No. WO 01/92309 relates to HBD3 and antimicrobial compositions containing it and methods of using same.
Mouse β-defensin 14 (MBD14) is an ortholog of HBD3 having a 68% sequence homology and similar spectrum of antimicrobial activity (Hinrichsen, K., et al., 2008, Rohrl, J. et al. 2008). MBD14 has chemotactic activity for HEK-293 cells (Soruri, A. et al., 2007) but its effects on CXCR4/SDF-1 interactions have not been determined.
The ability of HBD3 to modulate certain CXCR4/SDF-1 dependent activities implies that it may potentially affect HPC mobilization. However, complicated and costly synthesis, limited stability, and unknown toxicology and pharmacokinetics impair its therapeutic use, particularly systemically. Thus, the clinical effects of HBD3 in the mammalian system, particularly in the context of leukocyte mobilization and other therapeutic uses, remain to be investigated.
Hoover et al. (2003) examined several peptides derived from different regions of HBD3, including 36-38 amino acid (aa) fragments corresponding to carboxy terminal (C′) segments of HBD3, as well as shorter peptides in which the two cysteine residues were substituted for serine residues. Hoover et al. disclose that these peptides have anti-microbial properties, with different peptides having different target specificities.
Kluver et al. (2005) disclose certain other peptides and fragments of HBD3, including 27 and 40 aa C′ fragments of HBD3. These peptides varied in their antimicrobial activity as well as in their cytotoxicity to human hematopoietic cells and hemolytic activity, wherein some of these C′ fragments were highly toxic at 50 μM.
Krishnakumari et al. (2006) investigated the antibacterial activities of synthetic peptide analogs of human β-defensins, including a 22 aa peptide corresponding to the C′ of HBD3, wherein two cysteines were deleted.
WO 2007/126392 relates to isolated antimicrobial peptides which are linear analogs of HBD3, or fragments thereof. WO '392 teaches that the wild type (45-aa) HBD3 contains three disulfide, bonds (between positions 11 and 40, positions 18 and 33 and positions 23 and 40, respectively) which define its three dimensional folding; in the claimed peptides, one or more of the cysteine residues of HBD3 has been replaced by other amino acids or derivatives thereof, or by protected cysteine residues or derivatives thereof, or have been deleted, thereby removing these structural constrains. According to this publication, the linear backbone structure of HBD3 derivatives is a key structural determinant to decrease cytotoxicity to mammalian cells. WO '392 discloses that the claimed peptides may be used for inhibiting and/or reducing the growth of microorganisms, e.g. as medicaments for topical administration, eye drop compositions, contact lens solutions or medical device coatings. Among the disclosed sequences is a linear peptide derived from the C-terminus of wild type HBD3, corresponding to SEQ ID NO: 1 of the present invention.
None of the art discloses or suggests cyclic HBD3-derived peptides in which the N-terminus and the C-terminus of the peptide are linked through a peptide bond, or the use of β-defensin peptides as anti-cancer agents. Nor does the art teach or suggest that short peptides derived from HBD3 may be used effectively and safely for promoting HPSC mobilization and engraftment. There remains an unmet medical need for developing additional agents and protocols for cancer therapy and for providing improved bone marrow transplantation and recovery.